Fig 2: MiR-27a-3p downregulates GLP1R expression. (A) Schematic plot about miR-27a-3p targeting the 3' untranslated region of GLP1R and construction of dual luciferase. (B) Outcome of dual-luciferase assay for MC3T3-E cells treated with miR-27a-3p mimics. (C) Result of dual-luciferase assay for MC3T3-E1 cells treated with anti-miR-27a-3p. (D) GLP1R expression was examined. (E, F) Western blot was utilized to measure GLP1R expression. *p < 0.05 indicates a remarkable difference. NS: non-significant difference.

Fig 3: Expression of Ren1 four hours post single injection in mouse kidney. (A-L) Panel showing representative photos from Ren1 in situ hybridization and GLP-1R IHC double stain, from C57BL/6 J mice injected with a single dose of vehicle (top row), semaglutide (second row) or liraglutide (third row). DAPI nuclear stain (blue, first column), GLP-1R IHC stain labelled with FITC (green, second column), Ren1 ISH labelled with CY3 (yellow, column three), and merge of all three stains (Last column). Scale bars 100 µm, 40× magnification. (M) Quantification of Mm-Ren1 detection in renal vasculature from mice injected with vehicle (?), semaglutide (¦) or liraglutide (?). Individual values represent mean of 6–9 arteries. Data shown as individual values, mean and SEM. Analysed by Brown-Forsyth and Welch ANOVA followed by Games-Howell's test for multiple comparisons, ****p < .0001. (N) Dapb stain as negative control from vehicle injected (?), semaglutide injected (¦) and liraglutide injected (?) mice. Individual values represent mean of 6–9 arteries. Data shown as individual values, mean and SEM

Fig 4: Overexpressing GLP1R facilitates MC3T3-E1 pre-osteoblast differentiation and autophagy. (A, B) Protein expression of GLP1R. (C) mRNA expression of DMGs (Runx2, ALP, OCN, BSP, and Col1a1) in MC3T3-E1 cells. (D) qRT-PCR detected the mRNA expression of AMGs (LC3, ATG5, and ATG7) in MC3T3-E1 cells. (E, F) Western blot detected the protein expression of AMGs (LC3, ATG5, and ATG7). (G) LC3-II blot images about GFP-LC3 expression in MC3T3-E1 pre-osteoblasts and merged images of GFP-LC3 (green) and DAPI (blue). Figure A–G: Cells were grouped into miR-C + vector, miR-27a-3p + vector, and miR-27a-3p + oe-GLP1R, and then the treated cells were co-transfected into MC3T3-E1 pre-osteoblasts, followed by a quantitative detection after 24 h. *p < 0.05 denotes a remarkable significance.

Fig 5: GLP1 (glucagon-like peptide 1) receptor expression in human carotid bodies. A, GLP1R mRNA expression in human carotid bodies (CBs) assessed by RT-qPCR. Expression presented as Ct values in comparison to housekeeping (GAPDH) and reference (TH) genes. All reactions were performed on the same plate. TH – Tyrosine Hydroxylase; Boxplot hinges represent interquartile range (IQR=Q3–Q1). Red dot indicates an outlier (Q3+1.5*IQR). n=5. B and C, Localization of GLP1R in human CBs. GLP1R immunoreactivity (magenta) was detected in chemosensory glomus cells marked by UCHL1 (green). Arrows indicate blood vessels devoid of GLP1R immunoreactivity. Arrowhead indicates GLP1R-positive chemosensory glomus cell. n=1. Representative image selected best demonstrating GLP1R localization in a well-defined chemosensory glomus cell cluster. Scale bar—20 µm. D, Schematic model of GLP1 action on CBs in regulating sympathetic activity. Upon food ingestions, rise in blood glucose activates the CBs leading to sympathoexcitation and stimulates release of GLP1 from intestinal L-cells. GLP1 mediates insulin secretion which additively stimulates the CB chemoreceptors. GLP1 inhibits the chemosensory CB cells counteracting the sympathoexcitation to elevated levels of glucose and/or insulin in normal physiological context. Disruption of the GLP1 inhibitory component leads to aberrant sympathoexcitation as shown in the SH model. Treatment with GLP1R agonists act to reduce sympathetic activity by suppressing the peripheral chemoreflex drive from the CBs. TL/LEL indicates tomato lectin/lycopersicon esculentum lectin; and UCHL1, ubiquitin carboxy-terminal hydrolase L1.